Recombinant clusterin and use thereof in the treatment and prevention of disease

ABSTRACT

Recombinant clusterin polypeptides and compositions comprising the same are provided. In some aspects, recombinant clusterin or nucleic acids encoding the same may be used for treating and preventing an abnormality of morphology and function in a mammal with disease (e.g., cardiovascular diseases or alcoholism).

This application claims the benefit of U.S. Provisional PatentApplication No. 62/087,364, filed Dec. 4, 2014, the entirety of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSHP0306US_update_ST25.txt”, which is 60 KB (as measured in MicrosoftWindows®) and was created on Feb. 28, 2018, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates production of recombinantclusterin and the use thereof for prevention and treatment ofpathological conditions.

2. Description of Related Art

Hypercholesterolemia represents the most defined risk factor foratherosclerosis, an arterial disease that causes myocardial and cerebralinfarctions. Low density lipoprotein (LDL) carries cholesterol from theliver to peripheral tissues, and when elevated in blood, LDL depositsthe lipid in the arterial wall, which in turn develops atheroscleroticplaques and increases the risk for thrombogenic events in the arteries.In contrast, high density lipoprotein (HDL) functions as a reversecholesterol-transporter that removes the lipid from the arterial wall tothe liver where cholesterol is metabolized. In essence, LDL ispro-atherogenic while HDL anti-atherogenic (Nicholls et al., 2005;Ansell et al., 2004; von Eckardstein et al., 2005) LDL mainly containsApoB 100 and HDL apoE and apoA-I. In spite of the success of loweringLDL-cholesterol therapy with statins, raising HDL levels withtorcetrapib (an inhibitor of cholesterol ester transfer protein (CETP)has shown little benefit to patients with atherosclerosis (Kastelein etal., 2007; Nissen et al., 2007). The failure of torcetrapib therapyunderscores the incompleteness of our fundamental understanding of HDLfunction. HDL particles are heterogeneous in shape, density, size,composition and have multiple functional properties such as reversecholesterol transport, as well as anti-oxidant, anti-inflammatory, andanti-thrombotic activities. Indeed, dysfunctional, proinflammatory HDLhas been found in several pathological conditions, includingatherosclerosis (Smith et al., 2010), diabetes (Hoofnagle et al. 2010)and autoimmune disorders (McMahon et al., 2009; Volkmann et al., 2010).Thus, the development of an anti-atherogenic, anti-apoptotic, andanti-inflammatory agent that enables HDL beneficial action is ofclinical significance.

Clusterin is a sulfated, heterodimeric glycoprotein containing two 40kDa chains joined by a unique five disulfide bond motif 1). It containsseveral domains, such as amphipathic helix, heparin-binding domain, andlipid-binding domain. This protein was initially identified from ramrete testes fluid and named for its ability to elicit clustering ofSertoli cells supporting sperm maturation and development (NCBI/GenBankAccession No. NM_203339, NM_001831) Thereafter, species homologues havebeen isolated and cloned by a number of groups working in widelydivergent areas, generating various synonyms of clusterin, includingtestosterone repressed prostate message-2 (TRPM-2), sulfatedglycoprotein-2 (SGP-2), apolipoprotein-J (Apo-J), SP-40, 40, complementcytolysis inhibitor (CLI), and dimeric acidic glycoprotein (DAG), gp 80,NA1/NA2, glycoprotein III, etc.

Encoded on a 2-kb mRNA, clusterin is transcribed from a single copy genelocated on mouse chromosome 14 and human 8p21 (Fink et al., 1993), andtranslated as a 51 kD or so protein comprising 427 amino acid sequence(Jordan-Starck et al., 1994). In the blood stream, clusterin circulatesmainly with HDL as one of apolipoproteins (Choi-Miura et al., 1992;Stuart et al., 1992) but a small portion of clusterin may exist in LDL(Karlsson et al., 2005). Clusterin expression is induced by stressresponses (Wilson et al., 2000). Clusterin binds megalin/LRP-2 receptor,members of LDL receptor family. Increased expression of clusterin occursin both human (Mackness et al., 1997; Ishikawa et al., 2001; Ishikawa etal., 1998) and experimental animal (Jordan-Starck et al., 1994; Navab etal., 1997) atherosclerotic lesions. Reported functions of clusterininclude apoptosis inhibition (Kowolik et al. 2006), complement factorinactivation (Correa-Rotter et al., 1992), lipid recycling and transport(Gelissen et al., 1998), membrane protection, and maintenance ofcell-cell or cell-substratum contacts. It can effectively bind to lipidsand promote efflux of cholesterol and oxysterols from lipid-laden foamcells, a hall-marker of atherosclerosis (Gelissen et al., 1998).Clusterin has a high-affinity to a wide array of biological ligands. Thepresence of both hydrophilic and hydrophobic domains enables clusterinto act as a chaperone or a “biological detergent”.

Clusterin plays a role in regulation of metabolism and function ofvarious tissues and organs, particularly in the cardiovascular system.HDL with decreased levels of clusterin has been found in associationwith a high incidence of myocardiac infarction in patients withinsulin-resistant metabolic syndromes (Hoofnagle et al., 2010).Administration of an oral clusterin peptide was reported to reduceatherosclerosis in ApoE-null mice (Navab et al., 2005), and intravenousinjection of clusterin diminishes rat myocardiac infarction (Van Dijk etal., 2010). Transduction of clusterin can restore the mitochondrialmembrane potential and prevent the release of cytochrome-c frommitochondria into cytoplasma in cardiac myoblasts damaged by ethanol (Liet al., 2007). Furthermore, increased clusterin expression in myoblastsenhances the cell capacity of migration and homing through induction ofCXCR4, a chemokin-receptor for stromal cell-derived factor (SDF) (Li etal., 2010).

Human clusterin gene located in chromosome 8 (location 8p21-p18) with17876 by long contains 10 exons in total. Exon one and exon two arealternative yielding two different transcript isoforms. Other exons(Ansell et al., 2004; von Eckardstein et al., 2005; Kastelein et al.,2007; Nissen et al., 2007; Smith et al., 2010; Hoofnagle et al. 2010;McMahon et al., 2009; Volkmann et al., 2010) are shared with bothisoforms. clusterin transcripts contain 3 different translation startsites (ATG), all in-frame. The best characterized protein isoform isproduced from transcript isoform 2, where translation starts at thesecond ATG present in exon 2, right before ER-targeting signal.clusterin protein precursor (NP-976084) consists of 449 amino acids.There is evidence suggesting that two nuclear protein isoforms can beproduced from this transcript isoform, one in which translation startsat ATG in exon 3 (417 aa), and another with translation starting fromATG in exon 1 (459 aa). Secreted clusterin is produced from thetranscript isoform 2. The initial protein precursor, presecretory psCLU(˜60 kDa), becomes heavily glycosylated and cleaved in the ER, and theresulting alpha and beta peptide chains are held together by 5 disulfidebonds in the mature secreted heterodimer protein form, sCLU (˜75-80kDa).

Under stimulation by ionic radiation and oxidative stress, the nuclearclusterin is first translated as a non-glycosylated protein precursor,pnCLU (˜49 kDa), that is then translocated into nucleus. There isevidence of two distinct sized nuclear clusterin proteins (˜50 kDa and˜60 kDa) (Pajak et al., 2007), that could result from translationstarted either at ATG present in exon 3 or in exon 1, respectively.Secreted clusterin is cytoprotective but nuclear clusterin cytotoxic.The controversy of clusterin functions mainly results from the notwell-established role of the two different protein isoforms withdistinct subcellular localization and somewhat opposing functionalities.Some known functions include involvement in apoptosis through complexingwith Ku70 autoantigen (nCLU, proapoptotic) or interfering withBax-activation (sCLU, antiapoptotic) (Araki et al., 2005; Klokov et al.,2004; Leskov et al., 2003; Yang et al., 2000). Clusterin has also beenlinked to spermatogenesis, epithelial cell differentiation, TGF-betasignaling through Smad2/Smad3 (Shin et al., 2008; Ahn et al., 2008; Leeet al., 1992), complement activation (Dietzsch et al., 1992; O'Bryan etal., 1990). Secreted native clusterin contains the sequence domains ofnuclear clusterin critical for nuclear translocation and binding tonuclear death signaling proteins such as Ku70.

Despite the various roles in cellular regulation ascribed to clusterin,there remains a need for the development of recombinant clusterin andclusterin analogs as potential therapeutics. Embodiments of thisinvention disclose technology of producing recombinant clusterin with ahigh homology to the secreted form of native clusterin with a protectivefunction, and compositions of recombinant clusterin that can be used forprevention and treatment of diseases in a mammal.

SUMMARY OF THE INVENTION

In a first embodiment, a recombinant polypeptide is provided thatcomprises a mammalian clusterin coding sequence. In various aspects, theclusterin coding sequence may have a deletion of a nuclear localizationsignal and/or transmembrane domain (TMD). In some aspects, thepolypeptide may be a fusion protein comprising the clusterin codingsequence and a heterologous polypeptide sequence. For example, thepolypeptide may further comprise a tag sequence. In further aspects, thepolypeptide may comprise a protease cleavage site (e.g., a thrombincleavage site (Leu-Val-Pro-Arg-Gly-Ser; SEQ ID NO: 16) orenteropeptidase cleavage site (Asp-Asp-Asp-Asp-Lys; SEQ ID NO: 17)). Forexample, protease cleavage site can be positioned between the tagsequence and the clusterin coding sequence. In various aspects, the tagsequence may be a polyhistidine tag. In some aspects, the tag sequencemay be positioned N-terminally relative to the Clusterin codingsequence, while in other aspects the tag sequence may be positionedC-terminally relative to the Clusterin coding sequence.

In a further embodiment, a composition is provided that comprises aclusterin polypeptide of the present embodiments in a pharmaceuticallyacceptable carrier. In various aspects, the composition may be frozen orlyophilized.

In yet a further embodiment, an isolated polynucleotide molecule isprovided that comprises a nucleic acid sequence encoding a clusterinpolypeptide of the present embodiments. In some aspects, the nucleicacid sequence encoding the polypeptide may be operably linked to apromoter. In certain aspects, the promoter may be a promoter functionalin mammalian, bacterial or insect cells. In some aspects, thepolynucleotide molecule may be part of an expression vector, such as, aplasmid, an episomal expression vector or a viral expression vector.

In a further embodiment, a host cell is provided that comprises apolynucleotide molecule encoding a clusterin polypeptide of the presentembodiments. In some aspect, the host cell may be a bacterial cell, aninsect cell, or a mammalian cell. In some specific aspects, the hostcell is a human cell, such as a pluripotent cell, a cardiac cell, andendothelial cell, or a cardiac or endothelial precursor cell.

In yet a further embodiment, a method of manufacture a recombinantclusterin polypeptide is provided that comprises (a) expressing apolynucleotide molecule encoding a clusterin polypeptide of the presentembodiments in a cell; and (b) purifying the polypeptide from the cell.In various aspects, the polypeptide may comprise a purification tag, andpurifying the polypeptide may comprise use of a matrix having anaffinity for the purification tag. In some aspects, the purification tagmay be a polyhistidine tag, and purifying the polypeptide may comprisepurifying the polypeptide using a metal affinity column. In certainaspects, the purification tag may further comprise a protease cleavagesite positioned between the tag sequence and the clusterin codingsequence, and purifying the polypeptide may comprise contacting thepolypeptide with a protease that cleaves at the cleavage site.

In certain aspects, methods of the embodiments concern constructionand/or transfection of a nucleotide encoding clusterin into cells of amammalian cell or a non-mammalian cell causes sufficient expression ofclusterin polypeptides. In certain embodiments, the step of causing theexpression of an amount of a nucleotide encoding clusterin includestransfecting cells of the tissue with a DNA sequence encoding the entireclusterin polypeptide sequence, or a biologically active portion of theclusterin sequence, operably linked to a promoter and capable of beingexpressed in the cells to provide an amount of clusterin sufficient tobe identified, concentrated, extracted, and purified.

In still a further embodiment, a method of treating or preventing acardiovascular disease in a subject is provided that comprisesadministering an effective amount of a clusterin composition comprising(a) a recombinant clusterin polypeptide, (b) a polynucleotide (e.g., anexpression vector) encoding a clusterin polypeptide, and/or (c) cellsexpressing exogenous clusterin polypeptide of the present embodiments.In some aspects, the cardiovascular disease may be hypertension,hyperlipidemia, hypercholesterolemia, hyperglycemia, hypertension,atherosclerosis and atherosclerosis-associated ischemic heart failure,stenosis, calcification of cardiovascular tissues, stroke, myocardialinfarction or cerebral infarction. In some aspects, the cardiovasculardisease is hyperlipidemia, hypercholesterolemia or atherosclerosis. Instill further aspects, the cardiovascular disease may be diabetes. Invarious aspects, an effective amount of a clusterin composition may bean amount effective to reduce blood cholesterol, reduce blood glucose,reduce blood triglyceride, increase efflux of intracellular cholesterol,and/or increase vascular or cardiac cell survival. In some aspects aneffective amount of a clusterin composition provides enhancement orpromotion of cell survival and growth against cytotoxic or cytostaticfactors, including but not limited to, oxysterols, oxidizedlipoproteins, and proinflammatory cytokines.

In accordance with certain embodiments, a method of treating orpreventing atherosclerosis, or a complication thereof in a mammal, isprovided. For the purposes of this disclosure, the term “preventing”atherosclerosis has its usual meaning in the art and includes“deterring” and “reducing the risk of” atherosclerosis. This methodcomprises carrying out an above-described method wherein the tissue is acardiac or vascular or brain region comprising an atheroscleroticlesion, or an area that is at risk of forming an atherosclerotic lesion,and wherein the contacting of cells in the tissue with clusterinpolypeptides deters or prevents apoptotic cell death sufficiently toprevent, or reduce the risk of, formation of an atherosclerotic lesion.In some embodiments, the contacting of cells in the tissue withclusterin polypeptides deters or prevents tissue injury or degenerationsufficiently to prevent, or reduce the risk of, rupture of anatherosclerotic lesion.

In certain aspects, the atherosclerotic lesion comprises calcificationin a vessel or a valve with inflammation, which causes vascular tissuestiffness and cardiac or aortic valve malfunction, comprising stenosisor insufficient closure. In certain of the above-described methods, theamounts of clusterin compositions are effective to deter or preventcalcification and/or protect against inflammatory injury, induced by atleast one condition chosen from the group consisting of:hypercholesterolemia, hyperglycemia, hyperphosphatemia, and/orhypertension.

In some aspects, the atherosclerotic lesion or plaque comprises anunstable plaque caused by hyperlipidemia and inflammation, and theamount of clusterin contacting a treatment site are effective tostabilize the plaque (e.g., reduce the risk of rupture of the plaque,thrombus formation, or other complications).

Another embodiment of the present invention provides a method oftreating acute vascular syndromes and heart failure in a mammal, whichcomprises delivery of an amount of clusterin composition into the heartof the mammal to protect and improve heart function.

In yet a further embodiment, a method of treating or preventingalcoholism in a subject is provided that comprises administering aneffective amount of a clusterin composition comprising (a) a recombinantclusterin polypeptide, (b) a polynucleotide (e.g., an expression vector)encoding a clusterin polypeptide, and/or (c) cells expressing exogenousclusterin polypeptide of the present embodiments. In some aspects, theeffective amount of the clusterin composition is an amount effective toreduce withdrawal symptoms, alcohol intact or markers of liver damage ina subject.

In some aspects, a clusterin composition of the embodiments (e.g.,recombinant clusterin polypeptide, a polynucleotide encoding a clusterinpolypeptide, and/or cells expressing exogenous clusterin polypeptide)may be administered by intravenous injection or catheter delivery. Insome aspects, clusterin compositions are delivered into one or moretissue or organs of a mammal suffering from, or at risk of beingsubjected to, physical and/or chemical injury. In some embodiments thetissue is heart or vascular or brain tissue. In certain embodiments, thevascular or cardiac tissue is affected with atherosclerosis or heartfailure. In other embodiments, the vascular or cardiac tissue is notaffected with atherosclerosis, acute vascular syndromes, or heartfailure. In some embodiments, the cells are one or more of the celltypes: vascular endothelial cells, smooth muscle cells, cardiac myocytesand brain cells.

Thus, in certain aspects, there is provided a method of administering amammalian cell, such as a stem cell, with enhanced expression ofrecombinant clusterin, or that have been treated with recombinantclusterin, into a tissue or organ. In some embodiments, the tissue ororgan comprises a failing heart or an atherosclerotic blood vessel. Insome embodiments, the stem cells are administered by intravenousinjection, intra-arterial catheter, or by intramuscular or intratissueinjection. In certain embodiments, the stem cells are delivered orinjected together with an agent that causes vascular dilation and/or areco-administered with an anti-thrombotic agent. These and otherembodiments, features and advantages embodiments of the presentinvention will be recognized by those of skill in the art from thefollowing detailed description and drawings.

In further aspects, delivering an amount of recombinant clusterincomprises dissolving the polypeptide in a solution or buffer andinjecting the clusterin containing solution into blood stream or tissuesof a mammal or apply said clusterin solution on the surface of tissuesor organs with injury.

As used herein, the terms “clusterin”, “Apolipoprotein J”, and “Apo J”are used interchangeably.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%. In some aspects, most preferred is acomposition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—A schematic representation of the clusterin analog plasmidpCluAg. Representation of clusterin cDNA subcloning and construction ofan expression vector with an insert of clusterin analog-encoding cDNA isshown (SEQ ID NO: 26).

FIGS. 2A-2E—Polypeptide sequences and structures of various clusterinpolypeptides of the embodiments.

FIG. 3—Coomassie blue (G250) dye-stained polyacrylamide gel (SDS-PAGE)of recombinant human clusterin protein stored in solution and in drypowder for 3 months. The results show long term stability of thepolypeptides.

FIG. 4.—Immunoblotting analysis of the recombinant human clusterinpolypeptide. Clusterin polypeptides (1 μg/lane) were loaded intoSDS-PAGE (7%). After electropherosis, protein bands were transferredonto PVDF membrane, probed with rabbit anti-human clusterin antibodies(1:200), and immunostained bands were developed by chemiluminescence.The results again show long term stability of the polypeptides.

FIG. 5.—Ion exchange fast protein liquid chromatography (FPLC) ofrecombinant human clusterin proteins. Clusterin polypeptides wereseparated from other components in an aqueous solution, or buffer. Thebuffer flow rate was controlled by a positive-displacement pump and isnormally kept constant, while the composition of the buffer can bevaried by drawing fluids in different proportions from two or moreexternal reservoirs. Ion Exchange FPLC (BioRad UnoQ12 column); Buffer A:20 mM Tris (pH8.0) 0.5 mM EDTA; Buffer B: 20 mM Tris (pH8.0) 0.5 mMEDTA+1N NaCl; Flow rate: 2 ml/min; Injected amount: 250 ug; Result: peakeluted after 80 minutes.

FIGS. 6A-6C—Immunoblotting analysis of levels of recombinant clusterininjected intraveneously into the blood stream of wild type and ApoE-nullmice. Immunoblotting for clusterin and ApoAI proteins in the plasma ofWT and ApoE-null mice with different recombinant clusterin analog(CluAg) or saline control injection (7 days) (FIG. 6A). Densitometryshowed increased clusterin levels in Clu injected mice as compared tosaline injected one (FIG. 6B). Co-precipitation of clusterin (Clu) inthe plasma of C57BL/6 mice injected with recombinant human clusterinanalogs (FIG. 6C). Apolipoprotein-A1, a known HDL component, waspulled-down with anti-ApoA1 antibody, and Western blot analysis of theApoA1 pull-down was conducted with anti-clusterin antibodies.

FIG. 7—Immunoblotting analysis of clusterin and HisTR tag in proteinsextracted from the supernatants of human smooth muscle cell (SMC)cultures treated with oxLDL and recombinant clusterin analog (CluAg)with HisTR tag.

FIG. 8—Growth curves evidencing that incubation with recombinantclusterin analog stimulates proliferation of human vascular smoothmuscle cells. Human smooth muscle cells (SMC) were incubated in DMEMmedia containing CluAg-I (1-6 μg/ml).

FIG. 9—Growth curves evidencing that incubation with recombinantclusterin analog blocks inhibitory effect of oxidized lipoprotein andstimulates proliferation of human vascular smooth muscle cells (SMCs).Human SMCs were incubated in DMEM media containing CluAg-I (1-6 μg/ml)in the presence of 50 μg/ml oxidized low density lipoprotein (oxLDL).

FIG. 10—Blood plasma comparison of WT mice (upper panel) and ApoE^(−/−)mice (lower panel). Graphs show injection of human recombinantclusterin, CluAg-I, reducing blood levels of glucose, cholesterol,triglyceride, and LDL in ApoE^(−/−) atherosclerosis-prone mice but notin age and sex-matched normal wild type (WT) control mice.

FIG. 11—Blood pressure comparison of WT mice and ApoE^(−/−) mice. Graphsshow intravenous injection of human recombinant clusterin analog,CluAg-I, for 3 months reducing both systolic and blood pressure inApoE^(−/−) atherosclerosis-prone mice but has no effect on that in age(6-8 months old) and sex (male)-matched normal wild type (WT) controlmice.

FIG. 12—Echocardiography of normal wild type (WT) and ApoE−/− mice withCluAg-I injection. WT and ApoE^(−/−) mice were injected intravenouslywith CluAg for 3 months and then subjected to ultrasound examinationusing the Visualsonics™ echo device.

FIG. 13—Left ventricle ejection fraction comparison of WT mice andApoE^(−/−) mice. Intravenous injection of human recombinant clusterinanalog, CluAg-I, for 3 months reduces blood pressure in ApoE^(−/−)atherosclerosis-prone mice but not in age (6-8 months old) and sex(male)-matched normal wild type (WT) control mice.

FIGS. 14A-14D—Oil Red 0 staining of WT and ApoE^(−/−) aortas, evidencingthat weekly intravenous injection of human recombinant clusterin analog,CluAg-I, for 3 months reduces plaque sizes in ApoE^(−/−)atherosclerosis-prone mice, but not in age (6-8 months old) and sex(male)-matched normal wild type (WT) control mice. Reduced Oil red 0staining in ApoE−/− mice treated with CluAg is observed (FIG. 14D).

FIG. 15—Alizarin Red staining of murine vascular smooth muscle cellsevidence that CluAg reduces phosphate-induced calcification ofApoE^(−/−) SMCs in a matter dependent upon expression of ApoER2 andVLDLR. SMCs treated with ApoER2 or VLDLR shRNA were incubated with Pi(3.6 mmol/L)+/− CluAg in DMEM containing 5% FBS for 6 days. At the endof culture cells were washed in PBS, fixed in 10% formalin, and stainedin 2% Alizarin Red at 37° C. for 10 min. Reduced Alizarin Red stainingcould be partly blocked by ApoER2 and VLDLR shRNA.

FIGS. 16A-16C—Graphs show that CluAg treatment reduces phosphate-induced(Pi) calcification in ApoE^(−/−) vascular smooth muscle cells (SMCs) ina manner dependent upon expression of ApoER2 and VLDLR. SMCs treatedwith ApoER2 or VLDLR shRNA were incubated with Pi (3.6 mmol/L) in thepresence or absence of CluAg (6 μg/ml) in DMEM containing 5% FBS for 6days. In the end of culture cells were washed in PBS, incubated in 0.6MHCl overnight. Cell lysates were mixed with 0.1N NaOH was used to lysiscells and concentrations of proteins were measured. Supernatants werecollected, and calcium contents measured using a calcium assay; weremixed with 90 μl of the Chromogenic Reagent, and 60 μl of the CalciumAssay Buffer, mix gently; Incubated the reaction for 5-10 mins at roomtemperature, protected from light; Measured OD at 575 nm. 16A, SMCstreated with negative control ShRNA; 16B, with ApoER2 ShRNA; and 16C,with VLDLR ShRNA. Data represent means+/−S.D., **, p<0.01 and #p<0.05.

FIGS. 17A-17D—Alizarin Red staining showing that weekly intravenousinjection of human recombinant clusterin analog, CluAg for 3 monthsinhibits atherosclerosis as well as calcification in ApoE^(−/−)atherosclerosis-prone mice. 17A shows wild type (WT) control mice.Aortas were stained in 2% Alizarin Red at 37° C. for 10 min. Images weretaken using ×4 objective for 17A and 17B, and ×10 objective for 17C and17D. Reduced plaque size and Alizarin staining in ApoE−/− mice treatedwith CluAg was observed.

FIGS. 18A-18H—Immunofluorescence of bone morphogenic protein-2 (BMP2) inaortas of wild type (WT) mice and ApoE−/− mice treated with or withoutCluAg. Again, results show that CluAg treatment is able to reduce BMP2expression in ApoE−/− mice.

FIGS. 19A-19B—Calcium deposit in smooth muscle cells (SMC) isolated fromaorta of WT and ApoE-null mice. ApoE−/− VSMCs are more prone toPi-induced calcification. Alizarin red staining (19A) and calcium assay(19B) of VSMCs isolated from aorta of WT and ApoE−/− mice. Scale bar=200μm.VSMCs from WT and ApoE−/− mice were treated with inorganic phosphate(Pi) with or without Apo J (6 μg/mL). Bars represent means±SD, n=5,*P<0.05 vs. control; **P<0.01 vs. control; #P<0.05 vs. Pi group;##P<0.01 vs. Pi group.

FIGS. 20A-20B—Calcium deposit in SMC treating with or without Apo J.Inhibitory effect of Apo J on calcification in ApoE−/− VSMCs iscorrelated to concentration of Apo J. a: Alizarin S staining of VSMCsfrom ApoE−/− mice. VSMCs were treated with different concentrations ofApo J (0-12 μg/mL) during calcification. Scale bar=200 μm. b:Calciumassay of ApoE−/− VSMCs treated with various concentrations of Apo J(0-12 μg/mL) and Pi. Bars represent means±SD; n=5 per group. **P<0.01vs. control; ##P<0.01 vs. Pi group.

FIGS. 21A-21D—Apo J modulates the protein expression of smooth musclelineage-specific markers and calcification-related genes. VSMCs weretreated with Pi and Apo J (0-12 μg/mL). 21A: 40 μL cell culture mediumwas used to detect the level of Apo J in culture medium. Levels ofSM22α, αSMA and Runx2 protein expressions were assessed by western blot.GAPDH was used as the loading control. 21B-21D: Densitometry analysisshows the quantification of SM22α, αSMA and Runx2. Bars representmeans±SD; n=5 per group. *P<0.05 vs. control; **P<0.01 vs. control;#P<0.05 vs. Pi group; ##P<0.01 vs. Pi group.

FIGS. 22A-22B—Expression of Apo J during calcification. 22A: 40 μl cellculture medium was used to detect the expression of Apo J by Westernblot. 22B: Apo J mRNA expression was detected by qRT-PCR. GAPDH was usedas an internal control. Bars represent means±SD; n=3 per group. *P<0.05vs. control; **P<0.01 vs. control.

FIGS. 23A-23D—Apo J affects osteogenetic markers on mRNA levels duringcalcification. VSMCs were treated with Pi in combination with Apo J(0-12 μg/mL). Runx2 (23A), BMP-2 (23B), ALP (23C), and OPN (23D) mRNAexpressions were quantified by qRT-PCR and normalized to GAPDH as aninternal control. Bars represent means±SD; n=5 per group. **P<0.01 vs.control; #P<0.05 vs. Pi group; ##P<0.01 vs. Pi group.

FIGS. 24A-24F—Lentiviral shRNA knockdown of ApoER2 and VLDLR receptors.24A-24B: ApoE−/− cells were transfected with ApoER2 shRNA, VLDLR shRNAor negative control shRNA to collect whole-cell lysates for western blotanalysis of ApoER2 or VLDLR. 24C-24D: Densitometry analysis shows thequantification of ApoER2 or VLDLR expression. 24E-24F: Bar graphillustrating real-time PCR data demonstrating that ApoER2 or VLDLR wassuccessfully knocked down on mRNA level. Relative mRNA expressions werenormalized to GAPDH as an internal control. Bars represent means±SD; n=3per group. *P<0.05 vs. control, **P<0.01 vs. control; #P<0.05 vs. Pigroup, ##P<0.01 vs. Pi group.

FIGS. 25A-25B—Knockdown of ApoER2 or VLDLR gene abolishes the inhibitoryeffect of Apo J on calcification. Alizarin red staining (25A) andcalcium assay (25B) of receptor knockdown VSMCs under the treatment ofPi with or without Apo J (6 μg/mL). Scale bar=200 μm. Bars representmeans±SD; n=3 per group. **P<0.01 vs. control; ##P<0.01 vs. Pi group.

FIGS. 26A-26G—Knockdown of ApoER2 gene abolishes the effect of Apo J oncalcification markers and smooth muscle lineage-specific markers.ApoE−/− VSMCs were treated with Pi and Apo J (6 μg/mL). 26A: SM22α andRunx2 protein expressions in ApoER2 knockdown cells were detected bywestern blot. GAPDH was used as the loading control. 26A-C: Densitometryanalysis shows the quantification of SM22α and Runx2 in ApoER2 knockdowncells. 26D-G: Bar graph illustrating real-time PCR data showing the mRNAexpression of Runx2, BMP-2, ALP and OPN. Relative mRNA expressions werenormalized to GAPDH as an internal control. Bars represent means±SD; n=3per group. +P<0.05 means negative control shRNA infected cells vs.ApoER2 shRNA infected cells, ++P<0.01; *P<0.05 vs. control, **P<0.01 vs.control; #P<0.05 vs. Pi group, ##P<0.01 vs. Pi group.

FIGS. 27A-27G—Knockdown of VLDLR gene abolishes the effect of Apo J oncalcification markers and smooth muscle lineage-specific markers.ApoE−/− VSMCs were treated with Pi and Apo J (6 μg/mL). 27A: SM22α andRunx2 protein expressions in VLDLR knockdown cells were detected bywestern blot. GAPDH was used as the loading control. 27B-C: Densitometryanalysis shows the quantification of SM22α and Runx2 in VLDLR knockdowncells. 27D-G: Bar graph illustrating real-time PCR data showing the mRNAexpression of Runx2, BMP-2, ALP and OPN. Bars represent means±SD; n=3per group. +P<0.05 means negative control shRNA infected cells vs. VLDLRshRNA infected cells, ++P<0.01; *P<0.05 vs. control, **P<0.01 vs.control; #P<0.05 vs. Pi group, ##P<0.01 vs. Pi group.

FIGS. 28a-28d —ApoJ mutants with a peptide comprising athrombin-specific cleavage site exert an anti-coagulational effect bycompetitively inhibiting the conversion of fibrinogen into time of pigblood with (A) or without (B) ApoJ mutant. Fresh blood (0.3 ml) fromadult pig at different dilutions (0, ½, and ¼ in PBS) was mixed withrecombinant ApoJ mutant at 10 or 10 mg/ml), incubated at 37° C., andthen subjected to rotational thromboelastometry (ROTEM™). a, clottingtime; b, Alpha Angle clotting rapidity; c, maximum clot firmness; and d,maximum clot firmness at 10 minutes. Data represented from average oftwo separate experiments with the ApoJ mutant, Clusterin-ATMD-TRHis (SEQID NO: 8).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Clusterin is a multifunctional protein that may play an important rolein regulation of survival, proliferation and differentiation of adiversity of cell types in a mammal. The present disclosure providesrecombinant clusterin polypeptides and demonstrates the therapeuticefficacy of such recombinant polypeptides. In particular, data presentedherein demonstrates that recombinant clusterin can be effectivelysynthesized and purified and that the clusterin polypeptide preparationsare highly stable. The recombinant polypeptides appear to be essentiallynon-toxic, when administered to animals. However, in murine models forcardiovascular disease the clusterin polypeptides are able to reducemarkers of cardiovascular disease such as hyperglycemia, hypertensionand calcification as well as to normalize serum lipid levels. These dataindicate that the recombinant clusterin polypeptides of the embodiments(and cells and nucleic acids encoding the same) could be used as safeand effective therapeutics for treatment and prevention ofcardiovascular diseases and alcoholism.

I. Recombinant Clusterin Polypeptides

For the purposes of this disclosure, the terms “clusterin” or“recombinant clusterin” refers to proteins, whose sequence is based on amammalian clusterin sequence. In preferred aspects a recombinantclusterin polypeptide comprises a deletion of a nuclear localizationsignal, a transmembrane domain and/or is fused with a heterologouspolypeptide sequence (e.g., a purification tag). A skilled artisan willrecognize that deletions of the clusterin TMD likewise can disrupt theendoplasmic reticulum (ER)-targeting of recombinant clusterin. The terms“ApoJ” and “Clusterin” as used interchangeably herein. Examples ofspecific clusterin polypeptides include, without limitation,polypeptides provided as NCBI Acc. No. NM_203339, NM_001831, NM_013492,NM_053021, NM_012679, each of which is incorporated herein by reference.In certain aspects, the recombinant clusterin is about or at least about90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theclusterin polypeptide sequence of SEQ ID NOs: 1-3. For examples, in someaspects, the recombinant clusterin polypeptide about or at least about90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO:1, but comprises a deletion of a nuclear localization signal,ER-targeting sequence and/or a transmembrane domain. In yet furtheraspects, the recombinant clusterin is about or at least about 90%, 91%92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the clusterinpolypeptide sequence of SEQ ID NOs: 4-15.

Clusterin polypeptides and fragments, mutated, truncated or deletedforms of the clusterin and/or clusterin fusion proteins can be preparedfor a variety of uses, including but not limited to the generation ofantibodies, as reagents in diagnostic assays, as reagents in assays forthe identification of other cellular gene products involved in theregulation of clusterin mediated disorders, as reagents in assays forscreening for compounds that can be used in the treatment of clusterinmediated disorders, and as pharmaceutical reagents, useful in thetreatment of disorders related to clusterin.

Embodiments of the present invention also encompasses proteins that arefunctionally equivalent to the clusterin encoded by the nucleotidesequences described, as judged by any of a number of criteria, includingbut not limited to resulting in the biological effect of clusterin, achange in phenotype when the clusterin equivalent is present in anappropriate cell type. Such functionally equivalent clusterin proteinsinclude but are not limited to additions or substitutions of amino acidresidues within the amino acid sequence encoded by the clusterinnucleotide sequences described, but which result in a silent change,thus producing a functionally equivalent gene product.

In additional aspects, clusterin polypeptides may be further modified byone or more other amino substitutions while maintaining their biologicalactivity. For example, amino acid substitutions can be made at one ormore positions wherein the substitution is for an amino acid having asimilar hydrophilicity. The importance of the hydropathic amino acidindex in conferring interactive biologic function on a protein isgenerally understood in the art (Kyte and Doolittle, 1982). It isaccepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like. Thus such conservative substitution can be made in GrB andwill likely only have minor effects on their activity. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can beused as a guide and thus substitution of amino acids whosehydrophilicity values are within ±2 are preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. Thus, any of the GrB polypeptides describedherein may be modified by the substitution of an amino acid, fordifferent, but homologous amino acid with a similar hydrophilicityvalue. Amino acids with hydrophilicities within +/−1.0, or +/−0.5 pointsare considered homologous. Furthermore, it is envisioned that clusterinsequences may be modified by amino acid deletions, substitutions,additions or insertions while retaining its biological activity.

In some aspects, a clusterin polypeptide is fused with a heterologouspolypeptide sequence. For example, heterologous polypeptide sequencesmay be included to aid production or purification of a cell targetingconstruct. Some specific examples of amino acid sequences that may beattached to clusterin include, but are not limited to, purification tags(e.g., a T7, MBP. GST, HA, or polyHis tag), proteolytic cleavage sites,such as a thrombin or furin cleavage site, intracellular localizationsignals or secretion signals. In some aspects, a clusterin furthercomprises a cell-penetrating peptide (CPP). As used herein the terms CPPand membrane translocation peptide (MTP) as used interchangeably torefer to peptide sequences that enhance the ability of a protein to beinternalized by a cell. Examples for CPPs for use according to theembodiments include, without limitation, peptide segments derived fromHIV Tat, herpes virus VP22, the Drosophila Antennapedia homeobox geneproduct, protegrin I, as well as the T1, T2, and INF7 peptides.

Other mutations to the coding sequences described above can be made togenerate polypeptides that are better suited for expression, scale up,etc. in the host cells chosen. For example, the triplet code for eachamino acid can be modified to conform more closely to the preferentialcodon usage of the host cell's translational machinery, or, for example,to yield a messenger RNA molecule with a longer half-life. Those skilledin the art would readily know what modifications of the nucleotidesequence would be desirable to conform the nucleotide sequence topreferential codon usage or to make the messenger RNA more stable. Suchinformation would be obtainable, for example, through use of computerprograms, through review of available research data on codon usage andmessenger RNA stability, and through other means known to those of skillin the art.

Polypeptides corresponding to one or more portions of clusterin,truncated or deleted clusterin as well as fusion proteins in which thefull length clusterin or truncated clusterin is fused to an unrelatedprotein are also within the scope of the invention and can be designedon the basis of the clusterin nucleotide and clusterin amino acidsequences disclosed above. Such fusion proteins include but are notlimited to IgFc fusions which stabilize the clusterin polypeptide andprolong half-life in vivo or in in vitro assays; fusions to any aminoacid sequence that allows the fusion protein to be anchored to the cellmembrane; or fusions to an enzyme, fluorescent protein, or luminescentprotein which provide a marker function.

Additionally, the clusterin gene can be subcloned into a recombinantplasmid such that the gene's open reading frame is translationally fusedto an amino-terminal tag consisting of multiple (generally about six)histidine residues. Extracts from cells infected or transfected withsuch constructs are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

While clusterin polypeptides can be chemically synthesized (e.g., seeCreighton, 1983), large polypeptides derived from the clusterin and thefull length clusterin itself may advantageously be produced byrecombinant DNA technology using techniques well known in the art forexpressing nucleic acids. Such methods can be used to constructexpression vectors containing the clusterin nucleotide sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingClusterin nucleotide sequences may be chemically synthesized using, forexample, synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J., ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems can be utilized to expressthe clusterin nucleotide sequences of embodiments of the invention.Where clusterin polypeptide is a soluble derivative (e.g., with adeleted TMD), the polypeptide can be recovered from the culture, i.e.,from the host cell in cases where clusterin polypeptide is not secreted,and from the culture media in cases where clusterin polypeptide issecreted by the cells. However, the expression systems also encompassengineered host cells that express clusterin or functional equivalentsin situ, i.e., anchored in the cell membrane. Purification or enrichmentof clusterin from such expression systems can be accomplished usingappropriate detergents and lipid micelles and methods well known tothose skilled in the art. However, such engineered host cells themselvescan be used in situations where it is important not only to retain thestructural and functional characteristics of clusterin, but to assessbiological activity, e.g., in drug screening assays.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressclusterin sequences can be engineered, for example, as described in SEQID NOs: 4-15 and in the examples below. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines which express the Clusterin gene product.Such engineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of theClusterin gene product. A number of selection systems can be used,including but not limited to the herpes simplex virus thymidine kinase(Wigler, et al., 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962), and adenine phosphoribosyltransferase(Lowy, et al., 1980) genes can be employed in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler, et al., 1980; O'Hare, et al., 1981); gpt, whichconfers resistance to mycophenolic acid (Mulligan & Berg, 1981); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapin,et al., 1981); and hygro, which confers resistance to hygromycin(Santerre, et al., 1984).

The expression systems that can be used for purposes of the embodimentsinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing clusterinnucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing the clusterinnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing the Clusterinsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing Clusterin nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for theclusterin gene product being expressed. For example, when a largequantity of such a protein is to be produced, for the generation ofpharmaceutical compositions of clusterin protein or for raisingantibodies to the clusterin protein, for example, vectors which directthe expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited, to the E. coli expression vector pUR278 (Ruther et al., 1983),in which the clusterin coding sequence may be ligated individually intothe vector in frame with the lacZ coding region so that a fusion proteinis produced; pIN vectors (Inouye & Inouye, 1985; Van Heeke & Schuster,1989); and the like. pGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral; such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign sequences. The virusgrows in Spodoptera frugiperda cells. The clusterin gene coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofclusterin coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus, (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted polynucleotide is expressed (e.g., see Smith etal., 1983 and U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the clusterin nucleotide sequence of interest may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the clusterin gene product in infected hosts (e.g., See Logan& Shenk, 1984). Specific initiation signals may also be important forefficient translation of inserted clusterin nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire clusterin gene or cDNA, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of theclusterin coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBitter, et al., 1987).

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review, see Current Protocols in MolecularBiology, 1988; Grant, et al., 1987; Wu & Grossman, 1987; Bitter, 1987;and “The Molecular Biology of the Yeast Saccharomyces”, 1982.

In cases where plant expression vectors are used, the expression of thecoding sequence may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al., 1984), or the coat protein promoter of TMV(Takamatsu et al., 1987) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984; Broglie etal., 1984); or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al., 1986) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, for example, Methods for PlantMolecular Biology 1988; and Grierson & Corey, 1988.

In cases where an adenovirus is used as an expression vector, thenucleotide sequence of interest can be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene cam then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe gene product of interest in infected hosts (e.g., See Logan & Shenk,1984). Specific initiation signals such as those described above canalso be important for efficient translation of inserted nucleotidesequences of interest.

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38 and U937 cells.

II. Therapeutic Formulation and Administration

Therapeutics comprising clusterin polypeptides and/or nucleic acidsencoding clusterin polypeptides, such as those described in SEQ ID NOs:4-15 can be administered to a patient at therapeutically effective dosesof pharmaceutical preparations used to treat or ameliorate conditionssuch as, but not limited to, cardiovascular disease may be hypertension,hyperlipidemia, hypercholesterolemia, hyperglycemia, hypertension,atherosclerosis and atherosclerosis-associated ischemic heart failure,stenosis, calcification of cardiovascular tissues, stroke, myocardialinfarction or cerebral infarction. In some aspects, the cardiovasculardisease is hyperlipidemia, hypercholesterolemia or atherosclerosis. Instill further aspects, the cardiovascular disease may be diabetes. Invarious aspects, an effective amount of a clusterin composition may bean amount effective to reduce blood cholesterol, reduce blood glucose,reduce blood triglyceride, increase efflux of intracellular cholesterol,and/or increase vascular or cardiac cell survival. In some aspects aneffective amount of a clusterin composition provides enhancement orpromotion of cell survival and growth against cytotoxic or cytostaticfactors, including but not limited to, oxysterols, oxidizedlipoproteins, and proinflammatory cytokines, for examples those inducedby alcoholism. In further aspects a therapeutically effective doserefers to that amount of the compound sufficient to result in anyamelioration or retardation of disease symptoms or progression in amammal with acute vascular syndromes, to prevent and treat degeneration,stenosis and calcification of cardiovasulcar tissues, comprising valvetissues. In further aspects, clusterin derived polypeptides can be usedto prevent or treat male infertility by, for example, supporting spermmaturation and development, as has been described for native clusterin.

Toxicity and therapeutic efficacy of such clusterin derived compositionscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compositions which exhibit large therapeuticindices are preferred. While compositions that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat targets such compounds to the site of affected tissue in order tominimize potential damage to uninfected cells and, thereby, reduce sideeffects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compositions are preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compositionsused in the methods of the embodiments, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

When the therapeutic treatment of disease is contemplated, theappropriate dosage can also be determined using animal studies todetermine the maximal tolerable dose, or MTD, of a bioactive agent perkilogram weight of the test subject. In general, at least one animalspecies tested is mammalian. Those skilled in the art regularlyextrapolate doses for efficacy and avoiding toxicity to other species,including human. Before human studies of efficacy are undertaken, PhaseI clinical studies in normal subjects help establish safe doses.

Additionally, the bioactive agent (e.g., a clusterin polypeptide) may becomplexed with a variety of well-established compounds or structuresthat, for instance, enhance the stability of the bioactive agent, orotherwise enhance its pharmacological properties (e.g., increase in vivohalf-life, reduce toxicity, etc.).

Pharmaceutical compositions for use in accordance with the presentembodiments can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

The above therapeutic agents will be administered by any number ofmethods known to those of ordinary skill in the art including, but notlimited to, administration by inhalation; by subcutaneous (sub-q),intravenous (I.V.), intraperitoneal (I.P.), intramuscular (I.M.), orintrathecal injection; or as a topically applied agent (transderm,ointments, creams, salves, eye drops, and the like). Thus, thecompositions can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration can be suitably formulated to give controlled release ofthe active composition.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according tothe embodiments are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compositions can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions can also be formulated for rectal administration suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. The compositions may, if desired, be presentedin a pack or dispenser device which may contain one or more unit dosageforms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.

III. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—General Methods and Results

Preparation of native and recombinant clusterin analogs. To treat stemcells with clusterin, two forms of clusterin were prepared. Nativeclusterin (NCBI Accession No. NM_0134921, incorporated herein byreference). Clusterin was prepared from blood plasma using affinitychromatography with anti-clusterin antibody. In addition, a plasmid wasconstructed in which mouse or human clusterin cDNA is fused with aHis-tag and inserted under a promoter that will be activated in amammalian (e.g., human cells) or non-mammalian (e.g., bacteria) cell.Schematic presentation of the plasmid pCluAg with a clusterin cDNA-Hisinsert is shown in FIG. 1. pCluAg was constructed by insertingclusterin-TR/EK-His cDNA using a TOPO plasmid (Invitrogen). Recombinantclusterin produced by transfected mammalian cells (e.g., human 293cells) or bacterial cells (E. coli) was purified. Different clusterinanalog cDNAs were generated by RT-PCR and sequences predicting theencoding of different clusterin analog polypeptide sequences (FIGS. 2A-Dand Table 1, below).

TABLE 1 Sequence Descriptions SEQ ID Description: NO: CluAg-a(recombinant Clusterin) 1 CluAg-b (recombinant Clusterin-ΔTMD) 2 CluAg-c(recombinant Clusterin-ΔTMD-ΔNLS) 3 CluAg-Ia (HisTR-Clusterin) 4CluAg-Ib (HisTR-Clusterin-ΔTMD) 5 CluAg-Ic (HisTR-Clusterin-ΔTMD-ΔNLS) 6CluAg-IIa (Clusterin-TR-His) 7 CluAg-IIb (Clusterin-TR-His-ΔTMD) 8CluAg-IIc (Clusterin-TR-His-ΔTMD-ΔNLS) 9 CluAg-IIIa (HisEK-Clusterin) 10CluAg-IIIb (HisEK-Clusterin-ΔTMD) 11 CluAg-IIIc(HisEK-Clusterin-ΔTMD-ΔNLS) 12 CluAg-IVa (HisEK-Clusterin) 13 CluAg-IVb(HisEK-Clusterin-ΔTMD) 14 CluAg-IVc (HisEK-Clusterin-ΔTMD-ΔNLS) 15

Analysis of recombinant clusterin analog protein properties. The purityof recombinant clusterin analogs was examined by polyacrylamide gelelectrophoresis. Clusterin analog proteins are isolated from pCluAgtransfected cells, stored in solution or in dry powder, and loaded intoSDS-PAGE. After electrophoresis, fractions of proteins will beelectrotransferred onto a PVDF membrane, and probed with anti-clusterinantibodies. The membrane will be developed by using a chemiluminescencekit. Single bands of clusterin analog CluAg-I are visualized in SDS-PAGEstained with Commassia blue dye G250 (FIG. 3) and confirmed byimmunoblotting with anti-Clu antibody (FIG. 4). Furthermore, the analogswere examined by ion exchange chromatography, showing a clear eluent ofclusterin analogs (FIG. 5).

Clusterin analogs in the HDL fraction in mice. Apolipoprotein Edeficient (ApoE^(−/−)) mice are widely used murine mode foratherosclerosis. To evaluate the levels of clusterin association withApoAI in the blood, an immunoprecipitation method was developed withmonoclonal antibody against apoAI and clusterin. In the plasma of bloodfrom CluAg injected mice, ApoAI antibody co-precipitates ApoAI andCluAg, indicating that ApoAI is binding to CluAg (FIG. 6). To furtherconfirm the presence of CluAg and compare them to native clusterin,CluAg was added to SMC culture and incubated for 2 days with 5% serumcontaining native clusterin in the presence of oxLDL. Supernatants ofthe cultures were subjected to analysis of poly-His tag in CluAg. In theculture with or without oxLDL, CluAg apparently are free of the poly-Histag while CluAg (FIG. 7) in cell free cultures contains the poly-His tag(FIG. 7).

Clusterin analog regulation of vascular cell proliferation in culture.An in vitro system was first employed to examine the protective effectsof clusterin analogs on survival and growth of vascular cells. Cellswere treated with clusterin analogs at different concentrations in thecultures with or without oxidized LDL. After 2-4 days of stimulation,cell survival and proliferation were examined using a combination oftechniques including flow cytometry, fluorescent microscopy, andradioactive isotope labeling, as described below. Control experimentswere set up using other types of proteins, such as bovine albumin. FIGS.8 and 9 show growth curves of SMCs treated with or without CluAg (1-6μg/nl) in the presence or absence of oxLDL (50 μg/ml). Treatment withthe analog increased SMC proliferation even under the condition of oxLDLexposure. CluAg dose-dependently increases SMC proliferation even in thepresence of oxLDL (FIGS. 8 and 9).

Analysis of lipid profiles, cholesterol, glucose, and triglyceride inwild type and atherosclerosis prone mice injected with clusterin. Weeklyinjection of CluAg (30-40 μg) for 3 months was conducted in wild type(WT) and apoE-null mice. The blood samples were collected, during tailDNA sampling, from the mice injected with CluAg. Serum was prepared fromthe blood samples. Cholesterol levels, lipoprotein profiles andclusterin concentrations were determined respectively. In brief, serumdiluted in PBS was incubated in a 96 well plate coated with a rabbitpolyclonal antibody to clusterin. After incubation and washing in PBS,bound clusterin was detected by incubating with mouse monoclonalantibody to clusterin. Goat anti-mouse IgG conjugated with peroxidasewas used as the second antibody. Cholesterol and HDL was determined inthe laboratory of Department of Laboratory Medicine. The ratio ofclusterin vs. HDL was calculated after normalization with the lipidcontent. In addition to ELISA, immunoblotting assays were performed toverify the results. CluAg injection significantly reduced blood levelsof total cholesterol, glucose, triglyceride, and LDL in apoE-null micebut no changes were found in WT mice (FIG. 10).

Analysis of blood pressure. Blood pressure measurement was performed inthe tail artery using a tail-cuff method. Each measurement was repeated3 times to ensure reproducibility. Weekly injection of CluAg (30-40 μg)for 3 months led to reduction in the tail arterial blood pressure ofboth systolic and diastolic phases was conducted in apoE-null mice (FIG.11, left panel) but no changes in blood pressure were found in WT mice(FIG. 11, right panel).

Echocardiography of mice injected with CluAg. After weekly injection ofCluAg, morphological and functional changes were monitored usingechocardiography. B- and M-Mode echocardiography was performed one, 6,10, and 18 weeks after injection. The echocardiography studies wereconducted actually using ultrasonography as the murine heart is small.Mice were anesthetized with ketamine and xylazine, chests shaved and alayer of acoustic coupling gel will be applied to the thorax. Adynamically focused 9-MHz annual array transducer was applied using awarmed saline bag as a standoff. All echo studies were performed using astate of the art echo machine (HP Model Sonos 5500 HP). Area fractionswere determined by planimetry of diastolic and systolic volumes inparasternal short axis. The LV end-diastolic and end-systolic dimensionswere measured using the M-Mode from >3 beats by two independentinvestigators blinded to the research animals. LVEF (left ventricularejection fraction) was calculated as follows:LVEF=[(LVIDd)−(LVIDs)]/(LVIDd), LVIDd: end-diastolic left ventricularinternal diameter; LVIDs: end-systolic left ventricular diameter. FIG.12 shows ultrasound images of murine hearts with or without CluAginjection in both 2D and M-mode. FIG. 13 demonstrates that weeklyadministration of CluAg (30-40 μg/mouse) increases ejection fraction inthe hearts of ApoE−/− atherosclerosis-prone mice but no changes in wildtype mice with the same dose CluAg.

Oil red O staining of aortic wall. Mice were sacrificed 3 months afterCluAg treatment. Aortas were opened across the long axis and fixed in10% buffered formalin for histological evaluation. Aortas were stainedwith Oil Red O solution for assessing neutral lipid contents. Littlestaining was detected in WT aortas. However, compared to WT aortas, theApoE-null aortas were stained very intensively with Oil Red O, whichvisualizes atherosclerotic plaques. Decreased Oil Red O stains werefound in ApoE-null mice injected with CluAg protein (FIG. 14).

Analysis of aortic tissue and cell calcification. Aortic smooth musclecells (SMCs) were incubated with CluAg in the presence of sodiumphosphate (3.6 mmol/L) for 6 days. Alizarin red S (Sigma, St. Louis,Mo., USA) staining assesses calcium deposition in a reaction in whichAlizarin red S dye binds with Ca ions in cell layer matrix. Cells werefixed with 2% paraformaldehyde and stained with 1% Alizarin red S (pH4.2). The culture plates were photographed under a light microscope andassessed for the mineralized nodules which shown as red (FIG. 15). CluAgtreatment markedly reduces calcification in SMCs. To determine ifclusterin receptors mediate the inhibitory effect of CluAg on phosphateinduced calcification in SMCs, snRNA for ApoER2 and VLDLR, two knownreceptors for clusterin, was constructed and used to knock down ApoER2and VLDLR. CluAg treatment had no inhibitory effect on calcification inSMCs with the receptor knockdown with the snRNA (FIG. 15). This resultwas confirmed by direct calcium assays (FIGS. 16A-C). For assessing invivo calcification of aortic tissue, Alizarin red S staining wasperformed in the aortas of WT and apoE-null mice with CluAg injection.Sections of aortas from apoE-null mice but not wild type control miceshow the development of atherosclerotic plaques. Calcium deposits werehighly abundant in ApoE-null mice. Treatment with CluAg reducescalcification in ApoE-null mice (FIGS. 18A-H).

Immunohistochemistry of aortic tissue. In order to assess whether CluAginjection alters expression of calcification-regulatory proteins, suchas bone morphogenic protein (BMP)-2, in the aortic tissue, aorticsections were stained with anti-BMP-2 antibodies. Immunofluorescence forBMP-2 was developed with rhodamine-conjugated second antibodies (Sigma,St. Louis, Mo.). The slides were mounted in the Vectashield mountingmedium with 4′,6 diamidino-2-phenylindole (DAPI) (Vector, Burlingame,Calif.), and examined under an Olympus fluorescence microscope.Intensive BMP-2 immunofluorescence was found in ApoE-null aorta butCluAg injection reduced the intensity of the BMP-2 fluorescence.

Example 2—Further Analysis of Clusterin In Vivo

Methods and Materials

Cell culture. Aortic VSMCs were isolated from 5- to 6-week-old maleApoE−/− or WT mice with C57BL/6 background. The cells were maintained inDulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, Calif.,USA) supplemented with 10% fetal bovine serum (FBS) with 100 ng/mLpenicillin and streptomycin (Invitrogen) at 37° C. in a humidifiedatmosphere with 5% CO2. Calcification medium was made by adding NaH₂PO⁴(pH adjusted to 7.4) into 5% FBS medium to obtain a final concentrationof 3.6 mM inorganic phosphate. VSMCs from passage 5-10 were used. Apo JMedium was replaced every 2 or 3 days for up to 9 days. Cells maintainedin regular culture medium with 0.9 mM phosphate were used as controls.

Lentivirus infection and selection. To generate a stable (long-term)knockdown of ApoER2 or VLDLR gene expression in VSMCs, the VSMCs wereinfected with lentivirus particles containing a pLKO.1 vector with theinformation to express a shRNA against mouse ApoER2 or VLDLR. Thisplasmid also has a puromycin resistance gene, thereby allowing for theselection of cells stably expressing desired shRNA by addition ofpuromycin into culture medium. The optimal puromycin concentration forVSMCs before initiating the experiments (titration assay) was determinedto be 1 μg/ml. To perform lentivirus infection, cells approximately 80%confluent were used. 1.5 ml of fresh culture media containing virus wasadded onto cells. Fresh media was changed every 3 days 48 h afterinfection. To select the infected cells, selection media containingpuromycin was used for culture 48 h after infection. ApoER2 and VLDLRknockdown were monitored by western blot analysis and was achieved afterfour passages. One plate of cells infected with pLKO.1 vector with shRNAverified to contain no homology to known mammalian genes was maintainedin parallel. This plate served as a negative control for followingexperiments.

Calcium determination. Calcium content in VSMCs was determined bycolorimetric calcium detection kit (Abcam). Cells were washed with PBSand then incubated with 0.6M HCl under 37° C. overnight to bedecalcified. The calcium content in the supernatants was measured usingspectrometer. Then cells were lysed with 0.1 mol/LNaOH/0.1% SDS. Proteincontent was determined with BCA protein assay kit (Thermo Scientific)and calcium content was normalized to total protein content.

Alizarin Red S staining Cells were fixed in 2.5% glutaraldehyde andincubated with 2% Alizarin Red S under 37° C. for 15 minutes. Then VSMCswere rinsed with PBS three times. Calcium mineralization visualized byred staining was observed under microscope.

Real-time RT-PCR. Total RNA was extracted from VSMCs using Trizol(Invitrogen). 4 μg total RNA was used for cDNA synthesis in a reactionmixture of 204 with SuperScript III First-Strand Synthesis SuperMix(Invitrogen). Real-time PCR amplication was performed with IQTM SYBRGreen Supermix (Bio-Rad) in a ICYCLERIQ™ thermocycler (Bio-Rad). Thefollowing primers sets were used: ALP,5′-CACAATATCAAGGATATCGACGTGA-3′(sense; SEQ ID NO: 18) and5′-ACATCAGTTCTGTTCTTCGGGTACA-3′(antisense; SEQ ID NO: 19); BMP-2,5′-TTGTATGTGGACTTCAGTGATGTG-3′(sense; SEQ ID NO: 20) and5′-AGTTCAGGTGGTCAGCAAGG-3′ (antisense; SEQ ID NO: 21); Osteopontin,5′-TGGCTATAGGATCTGGGTGC-3′ (sense; SEQ ID NO: 22) and5′-ATTTGCTTTTGCCTGTTTGG-3′ (antisense; SEQ ID NO: 23); and Runx2,5′-TTACCTACACCCCGCCAGTC-3′ (sense; SEQ ID NO: 24) and5′-TGCTGGTCTGGAAGGGTCC-3′ (antisense; SEQ ID NO: 25).

Western blot. Proteins were isolated from VSMCs using RIPA buffer(Pierce) containing protease and phosphatase inhibitors (Sigma-Aldrich).Proteins separated on 10% SDS-Polyacrylamide gel were transferred topolyvinylidenedifluoride (PVDF) membranes (Millipore) and Western blotwas performed using the standard protocol. Membranes were blocked with5% bovine serum albumin (BSA) in TBS containing 0.1% Tween-20 (TBST).Primary antibodies were diluted in 3% BSA [goat anti-ApoJ 1:1000 (SantaCruz Biotechnology), rabbit anti-Runx2 1:1000 (Cell Signaling), goatanti-SM22α 1:5000 (Abcam), rabbit anti-αSMA 1:1600 (Abcam), rabbitanti-GAPDH 1:30000 (Abcam)] and were detected using HRP-conjugatedsecondary antibodies [donkey anti-goat 1:5000 (Santa CruzBiotechnology), goat anti-rabbit 1:20000 (Santa Cruz Biotechnology)]diluted in 3% BSA in TBST.

Data analysis and statistics. Western blot results were analyzed bydensitometry using Scion Image (Scion Corp). Real-time polymerase chainreaction data was quantified using EXCEL® software (Microsoft Corp).Values were graphed as mean±SD of at least triplicates determinations.Statistics (t test and ANOVA) were performed using Graphpad software(Graphpad. Software Inc). A value of P<0.05 was considered statisticallysignificant.

Results

Apo J attenuates calcification in both ApoE−/− and WT smooth musclecells. Because ApoE−/− mice are more prone to vascular calcificationthan WT ones, the responses of ApoE−/− and WT VSMCs to inorganicphosphate (Pi) were examined as well as the effect of Apo J oncalcification of these two groups of cells. The study shows that wheninduced with Pi, ApoE−/− VSMCs exhibited much higher levels ofcalcification on day 6 of treatment than WT cells. The addition of Apo J(6 μg/mL) into culture medium reduced calcium level in both groups, witha more dramatic inhibition on calcification in ApoE−/− VSMCs, shown byboth Alizarin S staining (FIGS. 19A and 20A) and calcium assay (FIGS.19B and 20B). Therefore, additional experiments were conducted usingApoE−/− VSMCs. Various concentrations (3 μg/mL, 6 μg/mL, 9 μg/mL, and 12μg/mL) of Apo J were then applied to test if the suppressive effect ofthe Apo J on vascular cell calcification is dose dependent. The datademonstrated that 6 μg/mL Apo J in culture is sufficient tosignificantly decrease calcium deposition level in ApoE−/− VSMCs, while12 μg/mL is incapable to further attenuate calcification compared to 9μg/mL group (FIG. 20B), possibly due to saturation of Apo J receptors.

Apo J modulates osteogenesis-related genes during calcification inApoE−/− VSMCs. To confirm the effect of Apo J on calcification, thechange of osteogenesis regulator Runx2 was assessed, as well as thesmooth muscle lineage markers SM22α and αSMA, with or without added ApoJ during calcification of ApoE−/− VSMCs, which were treated with Pi for6 days. Concentrations from 3 μg/mL to 12 μg/mL of Apo J were used.Equal volumes (40 μL) of culture medium were immunoblotted with Apo Jantibody at the endpoint of the experiment to verify the existence ofApo J protein in medium. The data suggested that both the mRNA level andthe native secreted form of Apo J increased in calcifying cells (FIGS.21A and 22A-B); addition of Apo J resulted in a much stronger signaldetected by Apo J antibody, indicating Apo J in medium was sustained ona potent level compared to control group and not subject to bulkdegradation through the period of experiment. It was found Runx2 wasincreased in response to Pi and this increase was attenuated in all ApoJ treated groups, with 6 μg/mL Apo J sufficient to keep Runx2 proteinexpression down to approximately the same level in uncalcified cells. Nosignificant differences of Runx2 levels were observed between 6 μg/mL, 9μg/mL, and 12 μg/mL groups (FIGS. 21A-21B). SM22α and αSMA proteinexpressions were downregulated by Pi compared to control group; Apo Jrescued the decreased level of SM22α and αSMA at a concentration assmall as 3 μg/mL (FIGS. 21A, 21C and 21D). mRNA expressions ofosteogenic genes including Runx2, BMP-2, OPN and ALP were increasedduring calcification while Apo J weakened this effect (FIGS. 23A-23D).

Knockdown of apolipoprotein-E receptor 2 (ApoER2 or very low densitylipoprotein receptor (VLDLR) abolishes the inhibitory effect of Apo J oncalcification. Because 6 μg/mL of Apo J has been shown to inhibitcalcium mineralization as well as activation of calcification-associatedgenes, this same concentration of Apo J was used to treat VSMCs inremaining experiments. Apo J receptors ApoER2 and VLDLR were knockeddown in ApoE−/− VSMCs by lentiviral shRNA. Knockdown effect wasconfirmed by western blots and qRT-PCR. The data demonstrated that theknockdown of VLDLR or ApoER2 itself didn't change calcification levelunder the influence of Pi. The negative control shRNA didn't alter thesensitivity of VSMCs to Apo J in calcification and calcium amount stilldecreased in Apo J treated negative control group. In contrast, VLDLR orApoER2 shRNA mediated knockdown of these receptors eliminated theremissive impact of Apo J on calcification, shown by Alizarin S andquantitative calcium assay (FIGS. 24A-24F), indicating Apo J functionsthrough ApoER2 and VLDLR to regulate calcification process.

Knockdown of ApoER2 or VLDLR partially abolishes the effect of Apo J onosteogenesis related genes in calcification. Knockdown of ApoER2 orVLDLR weakened the negative regulation of Apo J on the enhancedexpression of Runx2 in calcifying ApoE−/− VSMCs on protein level (FIGS.25A and 26C and mRNA level (FIG. 27C). SM22a protein expressiondecreased in Pi treated cells with ApoER2 or VLDLR knockdown despite thepresence of Apo J in culture medium (FIGS. 25B and 26D). Osteogenicmarkers alkaline phosphatase (ALP), bone morphogenic protein (BMP)-2,and Osteopontin (OPN) remained upregulated compared to uncalcifiedcontrol group in ApoER2 or VLDLR knockdown cells; no significantdifferences were found in mRNA expressions of these markers between ApoJ treated group and group without Apo J (FIGS. 27D-27F). This impliesthat knockdown of ApoER2 or VLDLR partially abolished the effect of ApoJ on osteogenesis related genes in calcification in ApoE−/− VSMCs.

Studies were undertaken to assess the effect recombinant Clusterin onblood coagulation. ApoJ mutants with a peptide comprising athrombin-specific cleavage site (e.g., Clusterin-TRHis,Clusterin-ATMD-TRHis, Clusterin-ATMD-ANLS-TRHis,HisTR-Clusterin-ATMD-ANLS, or HisTR-Clusterin-ATMD) exert ananti-coagulational effect by competitively inhibiting the conversion offibrinogen into time of pig blood with (A) or without (B) ApoJ mutant.Fresh blood (0.3 ml) from adult pig at different dilutions (0, ½, and ¼in PBS) was mixed with recombinant ApoJ mutant at 10 or 10 mg/ml),incubated at 37° C., and then subjected to rotational thromboelastometry(ROTEMTM). a, clotting time; b, Alpha Angle clotting rapidity; c,maximum clot firmness; and d, maximum clot firmness at 10 minutes. Datarepresented from average of two separate experiments with the ApoJmutant, Clusterin-ATMD-TRHis (SEQ ID NO: 8). Thus, these data indicatethat recombinant Clusterin with thrombin-cleavable peptide target, suchas Clusterin-TRHis, Clusterin-ATMD-TRHis, Clusterin-ATMD-ANLS-TRHis,HisTR-Clusterin-ATMD-ANLS, or HisTR-Clusterin-ATMD can exert ananti-coagulation effect by competitively inhibiting the conversion offibrinogen into fibrin and thus decreasing clot firmness and increasingthe clotting time of blood. Thus, recombinant Clusterin may serve as amulti-functional agent for the treatment of patients who suffer from aheart disease, such as heart disease complicated byhypercholesterolemia, hypertension, hyperglycemia and thrombogenesis.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A recombinant polypeptide comprising a Clusterincoding sequence, said coding sequence having at least 95% sequenceidentity to SEQ ID NO: 3, said sequence not including the Clusterinnuclear localization signal sequence (LEEAKKKK; SEQ ID NO: 27) and notincluding the Clusterin transmembrane domain sequence (LLLFVGLLL; SEQ IDNO: 28).
 2. The polypeptide of claim 1, comprising a Clusterin codingsequence having at least 98% sequence identity to SEQ ID NO:3.
 3. Thepolypeptide of claim 1, further comprising a heterologous polypeptidesequence fused to said Clusterin coding sequence.
 4. The polypeptide ofclaim 3, wherein the heterologous polypeptide sequence comprises aprotease cleavage site.
 5. The polypeptide of claim 3, wherein theprotease cleavage site is a thrombin cleavage site.
 6. The polypeptideof claim 1, wherein the polypeptide is aglycosylated.
 7. The polypeptideof claim 1, further comprising a tag sequence.
 8. The polypeptide ofclaim 7, further comprising a protease cleavage site positioned betweenthe tag sequence and the clusterin coding sequence.
 9. The polypeptideof claim 7, wherein the protease cleavage site is a thrombin orenteropeptidase cleavage site.
 10. The polypeptide of claim 7, whereinthe tag sequence is a polyhistidine tag.
 11. The polypeptide of claim 7,wherein the tag sequence is positioned N-terminally relative to theClusterin coding sequence.
 12. The polypeptide of claim 7, wherein thetag sequence is positioned C-terminally relative to the Clusterin codingsequence.
 13. A composition comprising a polypeptide of claim 1 in apharmaceutically acceptable carrier.
 14. An isolated polynucleotidemolecule comprising a nucleic acid sequence encoding a polypeptide ofclaim
 1. 15. A host cell comprising a polynucleotide molecule encoding apolypeptide of claim 1.